The S region of the murine histocompatibility complex (H-2) was defined almost 25 years ago as a genetic locus controlling the quantity of Ss (serum substance) in mouse serum. Subsequent work from a number of laboratories established that Ss is composed of two distinct proteins: C4, the murine fourth component of complement, and sex-limited protein (Slp), a protein that shares extensive structural and biochemical identity with C4 but which lacks C4 activity. Slp is notable in that its expression is testosterone-dependent and hence limited to males in some strains while in other strains expression is either independent of testosterone or conversely completely undetectable. Recent nucleic acid cloning and sequencing studies in our laboratory and others have demonstrated that C4 and Slp are encoded by distinct structural genes that lie in the S region and are doubtless the products of gene duplication. The goals of the proposed program are (a) to determine and compare the structural organization of the C4 and Slp genes; (b) to understand the structural and evolutionary relationships between these genes and other genes, in particular the class I and class II genes of the H-2 complex and the genes for the related complement proteins C3 and C5; and (c) to characterize the molecular mechanisms controlling sex-, tissue-, and strain- specific expression of the C4 and Slp genes. We propose to use recombinant DNA cloning and sequencing methods (a) to sequence the entire transcribed regions of the C4 and Slp genes; (b) to extend our study of upstream regions of the C4 and Slp genes by mapping restriction enzyme cleavage sites and by DNA sequencing; (c) to sequence regions upstream of the transcription initiation sites of C4 and Slp genes from mouse strains that exhibit varied C4 and Slp expression phenotypes; and (d) to use site-specific mutation and DNA reconstruction methods, together with methods for expression in cell culture to identify the gene segments responsible for regulating C4 and Slp expression.